CRP1, a LIM Domain Protein Implicated in Muscle Differentiation, Interacts with α-Actinin

Abstract

Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is alpha-actinin. We have shown that the CRP1-alpha-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1-alpha-actinin interaction is 1.8 +/- 0.3 microM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that alpha-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin-binding domain of alpha-actinin. In reciprocal mapping studies, we showed that alpha-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the alpha-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.

Full text


The Rockefeller University Press, 0021-9525/97/10/157/12 $2.00
The Journal of Cell Biology, Volume 139, Number 1, October 6, 1997 157–168
http://www.jcb.org
157
CRP1, a LIM Domain Protein Implicated in
Muscle Differentiation, Interacts with
a
-Actinin
Pascal Pomiès,* Heather A. Louis,* and Mary C. Beckerle*
*Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840
Abstract.
Members of the cysteine-rich protein (CRP)
family are LIM domain proteins that have been impli-
cated in muscle differentiation. One strategy for defin-
ing the mechanism by which CRPs potentiate myogen-
esis is to characterize the repertoire of CRP binding
partners. In order to identify proteins that interact with
CRP1, a prominent protein in fibroblasts and smooth
muscle cells, we subjected an avian smooth muscle ex-
tract to affinity chromatography on a CRP1 column. A
100-kD protein bound to the CRP1 column and could
be eluted with a high salt buffer; Western immunoblot
analysis confirmed that the 100-kD protein is
a
-actinin.
We have shown that the CRP1–
a
-actinin interaction is
direct, specific, and saturable in both solution and solid-
phase binding assays. The
K
d
for the CRP1–
a
-actinin
interaction is 1.8
6
0.3
m
M. The results of the in vitro
protein binding studies are supported by double-label
indirect immunofluorescence experiments that demon-
strate a colocalization of CRP1 and
a
-actinin along the
actin stress fibers of CEF and smooth muscle cells.
Moreover, we have shown that
a
-actinin coimmunopre-
cipitates with CRP1 from a detergent extract of smooth
muscle cells. By in vitro domain mapping studies, we
have determined that CRP1 associates with the 27-kD
actin–binding domain of
a
-actinin. In reciprocal map-
ping studies, we showed that
a
-actinin interacts with
CRP1-LIM1, a deletion fragment that contains the
NH
2
-terminal 107 amino acids (aa) of CRP1. To deter-
mine whether the
a
-actinin binding domain of CRP1
would localize to the actin cytoskeleton in living cells,
expression constructs encoding epitope-tagged full-
length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2
(aa 108-192) were microinjected into cells. By indirect
immunofluorescence, we have determined that full-
length CRP1 and CRP1-LIM1 localize along the actin
stress fibers whereas CRP1-LIM2 fails to associate with
the cytoskeleton. Collectively these data demonstrate
that the NH
2
-terminal part of CRP1 that contains the
a
-actinin–binding site is sufficient to localize CRP1 to
the actin cytoskeleton. The association of CRP1 with
a
-actinin may be critical for its role in muscle differenti-
ation.
Address all correspondence to Mary Beckerle, Department of Biology,
201 South Biology Building, University of Utah, Salt Lake City, UT 84112-
0840. Tel.: (801) 581-4485. FAX: (801) 581-4668. E-mail: beckerle@bioscience.
utah.edu
1.
Abbreviations used in this paper
: aa, amino acids; CEF, chicken embryo
fibroblasts; CRP, cysteine-rich protein; HBB, Hepes binding buffer; GST,
glutathione-S-transferase.
M
yogenesis
is a complex multistep process that
involves the specification of muscle progenitor
cells, the determination of a subset of these cells
to become myoblasts, the proliferation of these determined
cells, and ultimately the differentiation of these cells into
fully functional muscle. A variety of growth factors and
transcription factors, including members of the MyoD
family of basic helix–loop–helix proteins and the MEF2
family, contribute to the coordinated control of muscle cell
differentiation. These myogenic factors regulate both the
exit of myoblasts from the cell cycle as well as the initia-
tion of muscle-specific gene transcription (Cossu et al.,
1996; Molkentin and Olson, 1996). The ultimate product
of the muscle differentiation program is the ordered as-
sembly of an actomyosin-rich contractile machinery.
Recently, members of a family of proteins called the cys-
teine-rich protein (CRP)
1
family have been shown to be
involved in a late stage in muscle differentiation. In verte-
brates, the CRP family is comprised of three closely related
proteins, CRP1, CRP2, and the muscle LIM protein (MLP),
also referred to as CRP3 (Weiskirchen et al., 1995). CRP1
expression is prominent in smooth muscle derivatives and
is correlated with muscle development in avian embryos
(Crawford et al., 1994). Furthermore, overexpression of
either CRP1 or MLP/CRP3 potentiates the differentiation
of myoblasts in culture (Arber et al., 1994). Perturbation
of MLP/CRP3 expression by anti-sense RNA technology
results in a failure of muscle differentiation (Arber et al.,
1994). Similarly, elimination of MLP/CRP3 function in the
mouse by targeted gene disruption results in dramatic dis-
organization of myofibrils (Arber et al., 1997). Recently,

The Journal of Cell Biology, Volume 139, 1997 158
two
Drosophila
CRP family members,
Mlp60A
and
Mlp84B
,
have also been described (Arber et al., 1994; Stronach et
al., 1996). Both proteins exhibit muscle-specific expression
in developing embryos and a cytoskeletal localization
when expressed in vertebrate cells (Stronach et al., 1996).
Collectively, although the specific mechanism of action of
CRP family members is unknown, the available data sug-
gest a role for CRPs as essential positive regulators of
muscle differentiation.
Members of the CRP family exhibit a conserved molec-
ular architecture (Weiskirchen et al., 1995). CRPs exhibit
two tandemly arrayed LIM domains, each of which is
flanked by a conserved glycine-rich repeat (Weiskirchen
et al., 1995). The LIM domain is a cysteine-rich sequence
(CX
2
CX
16–23
HX
2
CX
2
CX
2
CX
16–21
CX
2
[C,H,D]) (Freyd et al.,
1990; Sadler et al., 1992) that coordinates two zinc atoms
(Michelsen et al., 1993) and mediates specific protein–pro-
tein interactions (Schmeichel and Beckerle, 1994). LIM
domains are found in a number of proteins that are in-
volved in control of gene expression and cell differentia-
tion. The LIM motif was first identified in three develop-
mentally regulated transcription factors,
Caenorhabditis
elegans
Lin-11, rat Isl-1, and
C. elegans
Mec-3, from which
the term LIM is derived (Freyd et al., 1990; Karlsson et al.,
1990). LIM domains can be found in association with func-
tional domains such as kinase domains, transcriptional ac-
tivation domains, or DNA-binding homeodomains. Alter-
natively, LIM domains sometimes represent the primary
sequence information in a protein.
In addition to their common structural features, CRP
family members are functionally related as well. CRP1 was
initially identified as a binding partner for zyxin, a low
abundance phosphoprotein that is concentrated at adhe-
sion plaques and in association with actin filament arrays
(Sadler et al., 1992; Crawford et al., 1994). All three CRP
family members have now been shown to bind directly to
zyxin (Louis et al., 1997). Moreover, all three proteins are
prominently associated with the actin cytoskeleton (this
report; Louis et al., 1997).
To understand the mechanism by which CRP1 affects
muscle differentiation, we have initiated an effort to iden-
tify CRP1-binding proteins in chicken smooth muscle, the
source from which CRP1 was originally purified (Craw-
ford et al., 1994). Here we report that CRP1 interacts di-
rectly with the actin-binding protein,
a
-actinin. Moreover,
we demonstrate that the two proteins are substantially
colocalized along the actin stress fibers. The findings re-
ported here suggest that CRPs may function as regulators
of myogenesis by virtue of their ability to interact directly
with
a
-actinin, an essential structural element in the myo-
fibril.
Materials and Methods
Isolation of Avian Smooth Muscle Proteins
Avian smooth muscle proteins were extracted from frozen chicken giz-
zards as described previously (Crawford and Beckerle, 1991; Crawford et
al., 1994). The resulting extract was sequentially precipitated with 27–34,
34–43, and 43–61% saturated ammonium sulfate. These ammonium sul-
fate precipitates were dialyzed against the column buffer (20 mM Tris-
acetate, pH 7.6, 0.1% 2-mercaptoethanol, 0.1 mM EDTA) before loading
onto affinity columns. The 27–34% ammonium sulfate precipitate con-
tains
a
-actinin whereas the 34–43% ammonium sulfate precipitate con-
tains CRP1.
Purification and Radioiodination of
a
-Actinin from
Avian Smooth Muscle
a
-Actinin was purified from the 27–34% ammonium sulfate precipitate as
described previously (Crawford et al., 1992). Cleavage of
a
-actinin by the
proteolytic enzyme thermolysin (Sigma Chemical Co., St Louis, MO) was
performed in 40 mM ammonium acetate, 1 mM CaCl
2
for 5 h at 20
8
C with
an enzyme to substrate ratio of 1:25. The
a
-actinin concentration was 3.2
mg/ml.
Purified
a
-actinin was radioiodinated as described previously (Craw-
ford et al., 1992), except that the incubation period of
a
-actinin with
[
125
I]Na was reduced to 2.5 min. The purity of the labeled
a
-actinin was as-
certained by SDS-PAGE followed by autoradiography.
Purification of Bacterially Expressed CRP1,
CRP1-LIM1, and CRP1-LIM2
CRP1-LIM1 corresponds to the NH
2
-terminal part of chicken CRP1
(cCRP1) (amino acids [aa] 1–107) including the NH
2
-terminal LIM do-
main followed by the first glycine-rich repeat of the protein. CRP1-LIM2
corresponds to the COOH-terminal portion of cCRP1 (aa 108–192) con-
taining the COOH-terminal LIM domain and the second glycine-rich re-
peat of the protein. Techniques for the purification of the bacterially ex-
pressed full-length CRP1 and CRP1-LIM2 were described previously
(Michelsen et al., 1993; Kosa et al., 1994). CRP1-LIM1 was purified in the
same manner as CRP1-LIM2 except that the CM-52 cation-exchange col-
umn was equilibrated in 5 mM potassium phosphate and 0.01% 2-mercap-
toethanol.
hCRP1 Expression, Isolation, and Radiolabeling
A plasmid engineered for the bacterial expression of human CRP1
(hCRP1) was generously provided by S.A. Liebhaber. The methods for
expression, purification and radiolabeling of the glutathione-S-transferase
(GST)-hCRP1 fusion protein were described previously (Feuerstein et al.,
1994). The GST–hCRP1 fusion protein was used in blot overlay assays
and in the solution binding assay whereas cCRP1 was used in all the other
experiments. There is a high degree of sequence similarity between hu-
man and chicken forms of CRP1 (91% identity; Crawford et al., 1994),
and the two proteins appear to be functionally interchangeable.
Affinity Chromatography
Bacterially expressed CRP1 or BSA was covalently coupled to Affi-gel 10
(Bio-Rad Laboratories, Hercules, CA) in coupling buffer (0.1 M Hepes,
pH 7.8, 0.1% 2-mercaptoethanol, 0.1 mM EDTA) for 4 h at 4
8
C. The
affinity resins were transferred to two different columns, washed with
coupling buffer, and then equilibrated with the column buffer (20 mM
Tris-acetate, pH 7.6, 0.1% 2-mercaptoethanol, 0.1 mM EDTA). A 27–34%
ammonium sulfate precipitate from avian smooth muscle was loaded onto
each column. The columns were washed with 0.1 M NaCl in column buffer.
Proteins eluted with 1 M NaCl in column buffer were collected in 300-
m
l
fractions and 15
m
l of each fraction were resolved by electrophoresis on
12.5% SDS–polyacrylamide gels.
a
-Actinin was detected by Western im-
munoblot analysis using a polyclonal antibody raised against chicken
a
-actinin that was generously provided by K. Burridge.
Gel Electrophoresis and Western
Immunoblot Analysis
SDS-PAGE was performed according to the method of Laemmli (1970)
except with 0.13% bisacrylamide. 12.5% polyacrylamide gels were used
routinely, however 17.5% gels were employed to resolve low mol wt pro-
teins such as CRP1-LIM1 and CRP1-LIM2. Western immunoblot analysis
was performed using horseradish peroxidase linked to protein A (Amer-
sham Life Science Inc., Cleveland, OH) as a second reagent and enhanced
chemiluminescent detection (Amersham Life Science Inc.).
Solution Binding Assay
GST-hCRP1 or GST agarose beads were incubated at 20
8
C with purified
a
-actinin or a 27–34% ammonium sulfate precipitate from avian smooth

Pomiès et al.
CRP1 Interacts with
a
-Actinin
159
muscle for 1.5 h on an orbital shaker. The agarose beads were washed
three times with PBS and three times with buffer B10 (20 mM Tris-ace-
tate, pH 7.6, 10 mM NaCl, 0.1 mM EDTA, 0.1% 2-mercaptoethanol). The
beads were then mixed in 40
m
l 2
3
Laemmli sample buffer (Laemmli,
1970), boiled, and the supernatants were analyzed by SDS-PAGE and
Western immunoblot using a polyclonal antibody raised against chicken
a
-actinin.
In competition experiments, GST-hCRP1 agarose beads were incu-
bated at 20
8
C with 100
m
l of [
125
I]
a
-actinin (500,000 cpm) for 1.5 h on an
orbital shaker in the absence of competing protein or in the presence of
unlabeled
a
-actinin or BSA. The agarose beads were washed three times
with PBS, centrifuged, and the counts bound to the agarose beads were
analyzed using a Packard Multi-Prias 1
g
counter (Packard Instrument
Co., Inc., Meriden, CT).
Blot Overlay Assay
Blot overlay assays were performed as previously described (Crawford et
al., 1992). Proteins were resolved by SDS-PAGE and transferred to nitro-
cellulose. The nitrocellulose strips were incubated in the presence of
[
32
P]GST or [
32
P]GST-hCRP1 fusion protein probes (600,000 cpm/ml), or
an [
125
I]
a
-actinin probe (250,000 cpm/ml). For competition experiments,
unlabeled competing proteins were added into the blot overlay buffer im-
mediately before the introduction of the labeled probe. Autoradiography
was performed at
2
80
8
C with an intensification screen.
Solid-phase Binding Assay
Removable microtiter wells (Dynatech Laboratories, Inc., Chantilly, VA)
were coated overnight at 4
8
C with 120
m
l of bacterially expressed CRP1 at
0.1 mg/ml. The wells were washed three times with Hepes binding buffer
(HBB) (20 mM Hepes, pH 7.4, 10 mM NaCl, 0.1 mM EGTA, 0.1% 2-mer-
captoethanol) and blocked with 300
m
l 2% BSA in HBB. After a 120-min
incubation at 37
8
C, the blocking solution was removed and the wells were
washed with HBB plus 0.2% BSA. The wells were next incubated for 2.5 h
at 37
8
C with [
125
I]
a
-actinin, in the presence of competing proteins in HBB.
The final volume was 120
m
l. At the end of the incubation period, the ra-
dioactive material was removed from the wells and they were washed six
times with HBB plus 0.2% BSA followed by a final rinse in HBB. The
wells were air dried and bound counts were determined using a
g
counter.
For these solid-phase binding studies, the
a
-actinin was radioiodinated to
a specific activity between 5.8
3
10
6
and 14.4
3
10
6
cpm/
m
g.
Confocal Immunofluorescence Microscopy
Chicken embryo fibroblasts (CEF) were cultured on glass coverslips in
DME supplemented with 10% FBS. Smooth muscle cells were prepared
from gizzards taken from 16-d-old chicken embryos as previously de-
scribed, except that trypsin was used instead of collagenase (Gimona et
al., 1990). Primary cultures derived from smooth muscle contained both fi-
broblasts and smooth muscle cells. We previously showed that differenti-
ated smooth muscle cells, which express the smooth muscle marker calpo-
nin, also exhibit dramatically higher levels of CRP expression than
fibroblasts and undifferentiated smooth muscle cells in the culture (Craw-
ford et al., 1994). Based on these observations, one can unequivocally
identify differentiated smooth muscle cells in the population based on
their CRP levels. Double-label indirect immunofluorescence (Beckerle,
1986) was performed using an anti–
a
-actinin primary monoclonal anti-
body (ICN Biomedicals Inc., Irvine, CA) followed by an FITC-conjugated
goat anti–mouse secondary antibody, and an anti–cCRP1 primary poly-
clonal antibody (B37) raised against the eleven carboxy-terminal amino ac-
ids GQGAGALIHSQ of cCRP1 followed by a Texas red–conjugated goat
anti–rabbit secondary antibody. The B37 antibody was generated by K.
Shepard and J.D. Pino in the Beckerle laboratory (University of Utah,
Salt Lake City, UT). All fluorochrome-labeled secondary antibodies were
obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove,
PA). Cells were viewed on a confocal laser scanning microscope (Bio-Rad
Laboratories, Hercules, CA) with an optical section height of 1
m
m.
Cell Labeling and Immunoprecipitation
CEF cells were radiolabeled with [
35
S]methionine-cysteine (Tran
35
S-label;
ICN Biomedicals Inc., Irvine, CA). Metabolic labeling was carried out
with adherent cells that were washed twice with PBS at 37
8
C and incu-
bated in one part DME, plus nine parts DME without methionine and
cysteine supplemented with 10% FBS in the presence of 200
m
Ci of
[
35
S]methionine-cysteine for 18 h. After three washes with PBS, the cells
were lysed in Laemmli sample buffer with protease inhibitors (0.1 mM
PMSF, 0.1 mM benzamidine, 1
m
g/ml pepstatin A, 1
m
g/ml phenantho-
line), and scraped off the dish. Cell lysates were boiled for 5 min. Immu-
noprecipitation was then performed as described below. In immunopre-
cipitation experiments using nonlabeled cells, smooth muscle cells from
adult chicken gizzards were lysed in 10 mM Tris, pH 8, 140 mM NaCl, 1%
Triton X-100, 0.2% deoxycholate, 0.02% SDS, 0.1 mM PMSF, 0.1 mM
benzamidine, 1
m
g/ml pepstatin A, 1
m
g/ml phenantholine, and scraped off
the dish. After incubation on ice for 30 min, the lysate was centrifuged at
10,000 rpm for 10 min, and the soluble material was recovered in the su-
pernatant. The supernatant was then incubated with protein A–agarose
beads (Sigma Chemical Co.) for 1 h at 4
8
C under gentle agitation. After a
2-min centrifugation at 2,000 rpm, the supernatant was incubated for 1 h
at 4
8
C with either 3
m
l of the polyclonal antibody B37 raised against CRP1
or 3
m
l of the corresponding preimmune serum, followed by a 1.5-h incu-
bation with protein A–agarose beads. At the end of the incubation period,
the beads were washed twice with the lysis buffer to remove the unbound
proteins; more extensive washing resulted in a loss of our ability to detect
protein that coimmunoprecipitated with CRP1. 40
m
l of 2
3
Laemmli sam-
ple buffer were then added to the pelleted beads and boiled for 5 min. The
immunoprecipitated proteins were resolved by SDS-PAGE. Gels contain-
ing metabolically labeled, immunoprecipitated proteins were dried and
subjected to autoradiography, while nonlabeled proteins were transferred
to nitrocellulose for immunoblotting as described above. CRP1 was de-
tected using the polyclonal antibody B37, while
a
-actinin was detected us-
ing the polyclonal antibody raised against chicken
a
-actinin provided by
K. Burridge.
Heterologous Expression and Immunofluorescence
Expression vector construction involved amplifying coding regions from
full-length cCRP1 cDNAs by PCR using Pfu Polymerase (Stratagene, La
Jolla, CA). Primers encoded EcoRV (5
9
end) or NotI (3
9
end) restriction
sites. Amplified fragments were digested and ligated into a pcDNA1/
NEO vector (Invitrogen, Carlsbad, CA) that was modified by inserting se-
quences encoding the myc epitope (EQKLISEEDLL) downstream from
the NotI site. Ligation at this site generated in-frame CRP1, CRP1-LIM1,
and CRP1-LIM2 fusions with myc. Constructs were sequenced prior to
use. Plasmid DNAs were isolated using a polyethyleneglycol precipitation
procedure (Sambrook et al., 1989) and were ultimately resuspended in
PBS for microinjection. Rat embryo fibroblast (REF52) cells were grown
to 50–70% confluence on coverslips in a 1:3 mixture of Ham’s F-12 and
DME containing 10% FBS, and microinjected with plasmid DNA at 250
ng/
m
l by a previously described technique in Beckerle and Porter (1983),
except an inverted microscope was used. Cells were fixed 24 h later and
processed for fluorescence microscopy with rhodamine-phalloidin (Mo-
lecular Probes, Inc., Eugene, OR) and indirect immunofluorescence
(Beckerle, 1986) with anti-myc primary monoclonal antibody and FITC-
conjugated goat anti–mouse secondary antibody (Jackson Immunore-
search Laboratories, West Grove, PA).
Results
Recovery of
a
-Actinin from a CRP1-affinity Column
Affinity chromatography was used to identify CRP1-bind-
ing proteins in an avian smooth muscle extract. Briefly,
proteins extracted from smooth muscle preparations were
fractionated by precipitation with increasing amounts of
ammonium sulfate (27–34, 34–43, and 43–61% saturation).
Each of the ammonium sulfate precipitates was subjected
to affinity chromatography on a CRP1 column. The CRP1
column was prepared from bacterially expressed avian
smooth muscle CRP1. We have shown previously that bac-
terially expressed CRP1 exhibits a native structure (Mich-
elsen et al., 1993, 1994) and retains the ability to bind zyxin
(Schmeichel and Beckerle, 1994). When a 27–34% ammo-
nium sulfate precipitate from the avian smooth muscle ex-
tract (Fig. 1
A
, lane
2
) is loaded on a CRP1 column, four
proteins of
z
115, 100, 41, and 35 kD elute from the column

The Journal of Cell Biology, Volume 139, 1997 160
with a high salt buffer as detected by silver staining (Fig. 1
B
, lane 2). By Western immunoblot analysis using specific
antibodies, we determined that the 100-kD protein that
binds to the CRP1 column is a-actinin (Fig. 1 B, lane 3).
No protein was detected using antibodies against the two
cytoskeletal proteins, talin and vinculin (data not shown).
Because zyxin has previously been shown to interact with
both a-actinin and CRP1 (Crawford et al., 1992; Sadler et
al., 1992), zyxin could theoretically have been responsible
for linking a-actinin to CRP1 in this experiment. How-
ever, no zyxin is detected in the 27–34% ammonium sul-
fate precipitate (Crawford and Beckerle, 1991), and there-
fore the CRP1-a-actinin interaction can not be mediated
by zyxin. In control experiments, the 27–34% ammonium
sulfate precipitate was loaded on a BSA column. In this case,
no a-actinin was recovered after a high salt buffer elution
as monitored by silver staining and Western immunoblot
(Fig. 1 B, lanes 4 and 5). Collectively, the results of these
experiments suggest that CRP1 can interact either directly
or indirectly with the actin binding protein a-actinin.
A Direct Interaction between CRP1 and
a
-Actinin Is
Detected under Nondenaturing Conditions
To examine whether CRP1 can interact directly with
a-actinin, we used an affinity resin-binding assay. Purified
a-actinin or a 27–34% ammonium sulfate precipitate con-
taining a-actinin (Fig. 1 A) was incubated with GST-CRP1
or GST (Fig. 1 C) coupled to glutathione-agarose beads.
After washing the beads, the proteins that remained
bound to the GST or GST-CRP1 affinity resins were re-
solved by SDS-PAGE. Western immunoblot analysis re-
vealed that GST-CRP1 (Fig. 1 D, lanes 4 and 6), but not
GST alone (Fig. 1 D, lanes 3 and 5), extracts a-actinin
from a solution of purified protein or from a complex mix-
ture of proteins. No band corresponding to a-actinin is de-
tected when GST-CRP1 agarose beads are incubated with
Figure 1. Specificity of the a-actinin–CRP1 interaction under
nondenaturing conditions. (A) A Coomassie blue–stained gel
showing molecular mass markers M, purified a-actinin (lane 1),
and the 27–34% ammonium sulfate precipitate from avian
smooth muscle extract (lane 2) that was loaded onto the affinity
columns and used in the affinity resin binding assay. (B) Lane 1,
Western immunoblot analysis of the 27–34% ammonium sulfate
precipitate that was loaded onto the affinity columns using a
polyclonal antibody raised against chicken a-actinin; lane 2, sil-
ver-stained gel showing the proteins eluted from the CRP1 col-
umn; lane 3, Western immunoblot analysis of the proteins shown
in lane 2 using a polyclonal antibody raised against a-actinin; lane 4,
silver-stained gel showing the material eluted from the BSA col-
umn; lane 5, Western immunoblot revealed that no a-actinin
bound to the BSA column (
a
-a, a-actinin). (C) Coomassie blue–
stained gel showing the purified GST (lane 1) and GST-CRP1
(lane 2) proteins that were used to generate the affinity resins.
(D) Western immunoblot analysis to detect chicken a-actinin.
The gel was loaded with a-actinin (lane 1) or a 27–34% ammo-
nium sulfate precipitate from a smooth muscle cell extract (lane
2). Purified a-actinin or proteins found in the 27–34% ammo-
nium sulfate precipitate were incubated with GST agarose (lanes
3 and 5) or GST-CRP1 agarose (lanes 4 and 6). a-Actinin binds
to the GST-CRP1 affinity resin. A mock affinity resin binding as-
say was performed with GST-CRP1 agarose beads in the absence
of a-actinin; no immunoreactive product is observed (lane 7). (E)
[
125
I]a-actinin was incubated with GST-CRP1 (left) or GST aga-
rose beads (right) in the absence of competing proteins (1
buffer), in the presence of a 2,000-fold molar excess of unlabeled
a-actinin (1 unlabeled a-actinin), or in the presence of an equiva-
lent molar amount of BSA (1 BSA). The counts bound to the
agarose beads were analyzed using a g counter and expressed as a
percentage of bound [
125
I]a-actinin in absence of competing pro-
teins. Mean and SEM from three experiments are shown.

Pomiès et al. CRP1 Interacts with
a
-Actinin 161
a solution that lacks a-actinin (Fig. 1 D, lane 7); this con-
trol confirms that detection of the immunoreactive 100-kD
protein is dependent on the addition of a-actinin and can-
not be due to crossreactivity of the antibody with fusion
protein dimers that migrate at a similar molecular mass.
These experiments show a direct interaction between CRP1
and a-actinin under nondenaturing conditions.
To examine the specificity of the CRP1–a-actinin inter-
action observed by the affinity resin-binding assay, we per-
formed a competition experiment (Fig. 1 E). [
125
I]a-Actinin
was incubated with GST-CRP1 or GST agarose beads in
the absence of competing proteins or in the presence of a
2,000-fold molar excess of either unlabeled a-actinin or
unlabeled BSA. The counts bound to the agarose beads
were measured using a g counter. The GST-CRP1 agarose
beads bind the radioiodinated a-actinin in the absence of
competing proteins or in the presence of BSA. In the pres-
ence of an excess of unlabeled a-actinin, binding of the ra-
diolabeled a-actinin to GST-CRP1 is reduced to the level
obtained with GST agarose beads. These results demon-
strate that the interaction between CRP1 and a-actinin is
direct, specific, and saturable under nondenaturing condi-
tions.
A Direct Interaction between CRP1 and
a
-Actinin Is
Detected by the Blot Overlay Assay
We have further characterized the CRP1–a-actinin inter-
action using a blot overlay assay that has been used previ-
ously to study many protein–protein interactions (Belkin
and Koteliansky, 1987; Crawford et al., 1992; Sadler et al.,
1992). We evaluated the ability of [
125
I]a-actinin to bind
directly to CRP1 present in fractions derived from an
avian smooth muscle extract. Three different ammonium
sulfate precipitates that include a diverse collection of
smooth muscle-derived proteins were resolved by SDS-
PAGE (Fig. 2 A) and transferred to nitrocellulose. CRP1
is found in the 34–43% ammonium sulfate (Fig. 2, lane 2)
but not in the 27–34 and the 43–61% ammonium sulfate
precipitates. Purified a-actinin was radioiodinated and
used as a probe to examine its ability to interact with CRP1
that was immobilized on nitrocellulose (Fig. 2 B). The pu-
rity of the [
125
I]a-actinin used in this experiment is shown
in Fig. 2 C. Among the proteins that are precipitated from
the smooth muscle extract, [
125
I]a-actinin recognizes a pro-
tein that exhibits an apparent molecular mass of 23 kD,
corresponding to the molecular mass of CRP1. A number
of other abundant proteins present on the nitrocellulose
membrane fail to interact with the radioiodinated a-actinin
showing the selectivity of the radiolabeled probe.
To analyze further the specificity of the CRP1–a-actinin
interaction, a competition experiment was performed us-
ing the blot overlay assay. Purified CRP1 was resolved by
SDS-PAGE (Fig. 3 A) and transferred to nitrocellulose.
The immobilized CRP1 was probed with radioiodinated
a-actinin in the absence or presence of a 2,000-fold molar
excess of unlabeled a-actinin or BSA (Fig. 3, B–D). The
radioiodinated probe interacts with the purified CRP1
confirming that, under the conditions of this experiment,
[
125
I]a-actinin interacts directly with CRP1. Moreover, in
the presence of unlabeled a-actinin, but not in the pres-
ence of an equimolar amount of unlabeled BSA, the bind-
ing of the radioiodinated a-actinin to CRP1 was dramati-
cally reduced. Collectively, these experiments demonstrate
that in the blot overlay assay, the association between
CRP1 and a-actinin is direct, specific and saturable.
The
a
-Actinin–CRP1 Interaction Displays a
Dissociation Constant in the Micromolar Range
The affinity of the association between a-actinin and
CRP1 was characterized by a solid-phase binding assay.
Purified bacterially expressed CRP1 (Fig. 3 A) was ad-
sorbed to microtiter wells, unoccupied sites on the plastic
wells were blocked with BSA, and the immobilized CRP1
was incubated with [
125
I]a-actinin. The amount of bound
[
125
I]a-actinin was determined by g counting. The specific-
ity of the CRP1–a-actinin interaction in this solid-phase
binding assay was evaluated by comparing the ability of
unlabeled a-actinin or BSA to compete with radiolabeled
a-actinin for binding to CRP1 (Fig. 4 A). A constant amount
of [
125
I]a-actinin was incubated in CRP1-coated wells in
the presence of increasing concentrations of competing
proteins. The interaction between CRP1 and [
125
I]a-actinin
is inhibited by the unlabeled a-actinin but not by an equiv-
alent molar excess of BSA, demonstrating the specificity
of the interaction between CRP1 and a-actinin in the
solid-phase binding assay. A typical curve predicted by the
simple binding reaction: CRP1 1 a-A ↔ CRP1?a-A, was
Figure 2. Demonstration of a direct interaction between CRP1
and [
125
I]a-actinin using a blot overlay assay. (A) Coomassie
blue–stained gel showing a 27–34 (lane 1), a 34–43 (lane 2), and a
43–61% (lane 3) ammonium sulfate precipitates from an avian
smooth muscle extract. Proteins from a parallel gel were trans-
ferred to nitrocellulose and the nitrocellulose strip was probed
with [
125
I]a-actinin. The resulting autoradiograph shown in B il-
lustrates [
125
I]a-actinin binding to CRP1. (C) Autoradiograph
demonstrating the purity of the radioiodinated a-actinin probe.
The position of the molecular mass markers is indicated on the
left, in kD.

The Journal of Cell Biology, Volume 139, 1997 162
obtained by plotting the moles of a-actinin bound to CRP1
against the concentration of free a-actinin (Fig. 4 B). Half
maximum binding in this experiment occurs at 1.9 mM free
ligand. From the average of three different experiments
using two different probes we calculate an average K
d
of
1.8 6 0.3 mM (mean 6 SEM) for the CRP1–a-actinin inter-
action.
CRP1 and
a
-Actinin Display Overlapping Subcellular
Distributions in CEF and Smooth Muscle Cells
The work described above reports the ability of CRP1 and
a-actinin to associate with each other in vitro. If this inter-
action also occurs in vivo, one might expect CRP1 and
a-actinin to be colocalized in cells. To examine this possi-
bility, we performed double-label immunofluorescence
microscopy using an anti-peptide antibody (B37) raised
against a sequence in cCRP1. By Western blot analysis of
a CEF lysate, the B37 antibody recognizes a single band
that exhibits an apparent molecular mass of 23 kD (Fig. 5
B) and comigrates with CRP1 (data not shown); no pro-
tein is detected using the preimmune serum (Fig. 5 B).
Similarly, a single band that migrates at an apparent mo-
lecular mass of 23 kD is immunoprecipitated from a deter-
gent extract of [
35
S]methionine–labeled CEF under dena-
turing conditions, whereas no immunoprecipitated band is
detected under the same conditions when the preimmune
serum is used (Fig. 5 C). The B37 antibody was used to
compare the subcellular distributions of CRP1 and a-acti-
nin using double-label indirect immunofluorescence in
CEF cells and in a primary culture of smooth muscle cells
from chicken gizzard (Fig. 6). By this approach, we ob-
serve that CRP1 and a-actinin are extensively colocalized
in cells along the actin stress fibers (Fig. 6, C and F), in ac-
cordance with the idea that they could interact in vivo. We
also observed that both proteins are present at the leading
edge of the cells, and in the adhesion plaques (Fig. 6 F).
However, in some adhesion plaques, where a-actinin is
present, CRP1 is not detected (data not shown). This ob-
servation is consistent with a previous report showing that
CRP1 is present in some adhesion plaques of CEF cells
but not in others (Crawford et al., 1994).
An In Vivo Interaction between CRP1 and
a
-Actinin in
Smooth Muscle Cells
We performed a coimmunoprecipitation experiment to
evaluate the ability of CRP1 to interact with a-actinin in
vivo. CRP1 can be immunoprecipitated from a smooth
muscle cell extract of smooth muscle cells under nondena-
turing conditions using the B37 anti–CRP1 antibody (Fig.
7 A). Under these conditions, a-actinin is detected in the
immunoprecipitate with CRP1 (Fig. 7 B), whereas another
cytoskeletal protein, vinculin, is not detected (data not
shown). Neither CRP1 nor a-actinin is detected when the
preimmune serum is used in the immunoprecipitation as-
say (Fig. 7, A and B). These data provide evidence that
a-actinin and CRP1 can be recovered as a complex from
smooth muscle cells.
Figure 3. Specificity of the [
125
I]a-actinin–CRP1 interaction. (A)
Coomassie blue–stained gel showing molecular mass markers and
the purified recombinant CRP1. Autoradiograph of parallel ni-
trocellulose strips probed with [
125
I]a-actinin in the absence of
competing protein (B), or in the presence of either a 2,000-fold
molar excess of unlabeled a-actinin (C), or a 2,000-fold molar ex-
cess of unlabeled BSA (D).
Figure 4. Binding of [
125
I]a-
actinin to CRP1 in a solid-
phase binding assay. (A) The
specificity of the association
between CRP1 and [
125
I]a-
actinin in a solid-phase bind-
ing assay was analyzed in a
competition experiment. A
constant amount of [
125
I]a-
actinin (0.26 pmoles in 120
ml) was incubated in CRP1-
coated wells with increasing
concentrations of unlabeled
a-actinin (1 a-actinin) or
BSA (1 BSA). In this exper-
iment, a total of 3,076 cpm
were bound specifically to
CRP1 when no competing
unlabeled a-actinin was
added. The data are ex-
pressed as a percentage of
the maximum binding obtained when the [
125
I]a-actinin is incu-
bated with the CRP1-coated wells in the absence of competing
protein. (B) From the competition experiment shown in A, the
moles of a-actinin bound to CRP1 were plotted as a function of
the free a-actinin concentration. In this particular experiment,
the a-actinin was radioiodinated to a specific activity of 5.8 3 10
6
cpm/mg; assuming a mol wt of 200,000 g/mol for a-actinin. The
calculated dissociation constant (K
d
) was 1.9 mM. The mean dis-
sociation constant determined from three different experiments
using two different probes is 1.8 6 0.3 mM (mean 6 SEM).

Pomiès et al. CRP1 Interacts with
a
-Actinin 163
Mapping the Domains of CRP1 and
a
-Actinin That
Participate in the Interaction of the Two Proteins
To map the binding site for CRP1 on a-actinin, we per-
formed a blot overlay experiment using labeled CRP1.
a-Actinin can be separated into two well-characterized
proteolytic products of 53 and 27 kD by cleavage with
thermolysin. The 27-kD fragment has been shown to inter-
act with zyxin, vinculin, and actin, whereas the 53-kD frag-
ment is essential for dimerization of the protein and for
interacting with the cytoplasmic domain of b
1
integrin re-
ceptors for extracellular matrix (Mimura and Asano, 1986;
Otey et al., 1990; Pavalko and Burridge, 1991; Crawford et
al., 1992). Fig. 8 A shows a Coomassie blue–stained gel of
purified a-actinin and the products of partial proteolytic
cleavage with thermolysin. Proteins from a parallel gel
were transferred to nitrocellulose and the resulting blot
was incubated with [
32
P]GST-CRP1 (Fig. 8 B). By this blot
overlay approach, [
32
P]GST-CRP1 associates directly with
a-actinin and also prominently with the 27-kD actin-bind-
ing domain of a-actinin; no interaction of CRP1 with the
53-kD fragment is observed. When [
32
P]GST is used as a
probe, no detectable protein binding is observed (Fig. 8
C). The purity of the
32
P-labeled probes used in this exper-
iment is shown in Fig. 8 D. These results demonstrate that
the binding site for CRP1 on a-actinin is in the 27-kD actin-
binding domain of a-actinin.
CRP1 displays two LIM domains separated by 56 amino
acids (Crawford et al., 1994). To characterize the binding
site for a-actinin on CRP1, we compared the ability of
a-actinin to interact with full-length CRP1 and two pep-
tides, CRP1-LIM1 and CRP1-LIM2, derived from the in-
tact protein. CRP1-LIM1 corresponds to the NH
2
-termi-
nal part of CRP1 (aa 1–107) containing the NH
2
-terminal
LIM domain followed by the first glycine-rich repeat of
the protein, and CRP1-LIM2 corresponds to the COOH-
terminal part of the protein (aa 108–192) containing the
COOH-terminal LIM domain and the second glycine-rich
repeat. CRP1, CRP1-LIM1, and CRP1-LIM2 were re-
solved by SDS-PAGE (Fig. 9 A), were transferred to ni-
trocellulose and were probed for their ability to interact
with [
125
I]a-actinin in a blot overlay assay (Fig. 9 B). The
radioiodinated a-actinin interacts with the bacterially ex-
pressed purified CRP1 and with CRP1-LIM1. The molar
amounts of the two single LIM peptides, CRP1-LIM1 and
CRP1-LIM2, loaded on the gel was twice the amount
loaded for the double LIM protein, CRP1. Although the
[
125
I]a-actinin bound only to intact CRP1 and the CRP1-
LIM1 peptide, the binding to the deletion construct
reached only about 50% of the binding observed with full-
length CRP1, as measured by PhosphorImager analysis
(data not shown). No interaction is detected between the
[
125
I]a-actinin and CRP1-LIM2. Thus it appears that the
CRP1-LIM1 peptide contains sequence information that
establishes a docking site for a-actinin; the generation of
the CRP-LIM1 truncation may have rendered the a-actinin
binding site suboptimal. We cannot rule out the possibility
that other low affinity binding sites for a-actinin exist in
CRP1, however, the only site we have been able to map is
within the CRP1-LIM1 region.
Colocalization of CRP1 and CRP1-LIM1
with Actin Filaments
Given the fact that a-actinin interacts with CRP1-LIM1,
the NH
2
-terminal part of CRP1, we examined the possibil-
ity that the CRP1-LIM1 peptide contains sequence in-
formation involved in targeting the protein to the actin
cytoskeleton. Eukaryotic expression constructs encoding
epitope-tagged full-length CRP1, CRP1-LIM1, and CRP1-
LIM2 were microinjected into cells. We used double-label
indirect immunofluorescence to compare the subcellular
distributions of the expressed portions of CRP1 and the
actin stress fibers. The expressed CRP1 is associated with
the actin cytoskeleton (Fig. 10, A and B); this localization
corresponds to the typical distribution of endogenous
CRP1 in fibroblasts (Sadler et al., 1992; Crawford et al.,
1994). The CRP1-LIM1 peptide also localizes with F-actin
(Fig. 10, C and D) whereas expressed CRP1-LIM2 fails to
associate with the actin cytoskeleton (Fig. 10, E and F).
We detected some nuclear localization of the two deletion
constructs, CRP1-LIM1 and CRP1-LIM2; however, the
significance of this finding is not clear. Some expressed
protein is found in a punctate pattern that does not corre-
spond to the distribution of filamentous actin; because we
do not observe such a prominent punctate pattern when
we visualize endogenous CRP1 by indirect immunofluo-
rescence, the physiological relevance of this distribution is
questionable. These heterologous expression studies in rat
embryo fibroblasts reveal that deletion of aa 1–107 from
CRP1 eliminates the protein’s ability to localize to the ac-
tin cytoskeleton. The NH
2
-terminal 107 aa of CRP1 re-
tains the capacity to localize to the cytoskeleton illustrat-
Figure 5. Characterization of an anti-peptide antibody (B37) di-
rected against cCRP1. (A) A Coomassie blue–stained gel show-
ing molecular mass markers M and total CEF proteins L. (B) A
parallel gel was transferred to nitrocellulose and probed with the
anti-CRP1 antibody B37 or its corresponding preimmune serum
pre. A single polypeptide of 23 kD is recognized by the antibody.
(C) Autoradiograph of a gel loaded with a CEF lysate prepared
from [
35
S]methionine-cysteine–labeled cells L, the proteins im-
munoprecipitated from this lysate with the polyclonal antibody
raised against CRP1 B37, and with its corresponding preimmune
serum pre. A single protein of 23 kD is specifically immunopre-
cipitated with the antibody against CRP1.

The Journal of Cell Biology, Volume 139, 1997 164
ing that this region is both necessary and sufficient to
support the cytoskeletal association of CRP1.
Discussion
In this study, we have identified the actin-binding protein,
a-actinin, as a new binding partner for CRP1, a LIM do-
main protein that has been implicated in the process of
muscle differentiation. We have used a variety of solution
and solid-phase binding assays to demonstrate and charac-
terize an association between a-actinin and CRP1. By
these approaches we have shown a direct, specific, and sat-
urable interaction between a-actinin and CRP1. Because
both smooth muscle and bacterially expressed CRP1 inter-
act with a-actinin, eukaryotic posttranslational modifica-
tion of CRP1 is not required for binding of these two pro-
teins. From our solid-phase binding studies, the interaction
between a-actinin and CRP1 appears to occur at a single
site with a z1.8 mM K
d
, corresponding to a moderate af-
finity interaction between the two proteins. The dissocia-
tion constant values calculated for the interactions be-
tween a-actinin and its other binding partners are in the
same range (Fig. 11 A). The biochemical studies that pro-
vide evidence for an interaction between a-actinin and
CRP1 are supported by immunocytochemical studies that
demonstrate that the primary distributions of a-actinin
and CRP1 in CEF and smooth muscle cells are very simi-
lar, with both proteins prominently concentrated along the
actin cytoskeleton and to a more limited extent within
adhesion plaques. Although some immunostaining of cell
nuclei is evident with anti-CRP antibodies and some accu-
mulation of the proteins within nuclei is observed in over-
expression studies, the physiological relevance of the
nuclear CRPs is not clear. CRPs are relatively small pro-
teins that would not be excluded from nuclei based on size.
If CRP did diffuse into the nucleus of a cell, it might be
Figure 6. CRP1 and a-actinin are extensively codistributed in CEF and in smooth muscle cells. CEF cells (A–C) and smooth muscle
cells (D–F), prepared for confocal indirect immunofluorescence microscopy, were double-labeled with a polyclonal antibody raised
against CRP1 (A and D), and a monoclonal antibody raised against a-actinin (B and E). C and F are composite images of CRP1 (green)
and a-actinin (red) staining; the overlapping regions appear in yellow. Confocal microscopy reveals that CRP1 and a-actinin are exten-
sively colocalized along the actin stress fibers. Both a-actinin and CRP1 are detected at the leading edges of the cells (arrows) and in the
adhesion plaques (arrowheads and data not shown). Bars, 30 mm.
Figure 7. An in vivo interac-
tion between CRP1 and
a-actinin in smooth muscle
cells. Proteins were immuno-
precipitated from a chicken
gizzard smooth muscle ly-
sate L with the polyclonal an-
tibody raised against CRP1
B37 and with the correspond-
ing preimmune serum pre.
The immunoprecipitated pro-
teins were resolved by SDS-
PAGE and were transferred
to nitrocellulose and probed
with polyclonal antibodies
raised against CRP1 (A) or
a-actinin (B). a-actinin is im-
munoprecipitated under nondenaturing conditions with the anti-
CRP1 antibody, but not with the preimmune serum. The position
of the molecular mass markers is indicated on the left in kD.

Pomiès et al. CRP1 Interacts with
a
-Actinin 165
passively trapped there as a result of its basic nature; the
isoelectric point of CRP1 is 8.5 (Crawford et al., 1994).
That said, it is not possible to rule out a nuclear function
for CRP1; this possibility remains intriguing since the
three-dimensional conformation of a LIM domain derived
from CRP1 exhibits features that would be expected to be
compatible with nucleic acid binding (Perez-Alvarado et
al., 1994). Moreover, Drosophila CRPs are not excluded
from cell nuclei in the developing musculature, in contrast
with myosin which is clearly excluded from the nuclear
compartment in the same cells (Stronach et al., 1996).
a-Actinin has been extensively studied and much is under-
stood about its biochemical properties. a-Actinin forms
antiparallel homodimers (Wallraff et al., 1986; Imamura et
al., 1988) that cross-link actin filaments into parallel arrays
(Maruyama and Ebashi, 1965; Podlubnaya et al., 1975). In
nonmuscle cells such as cultured fibroblasts, a-actinin is
found along the stress fibers and in the adhesion plaques
where actin filament bundles associate with the plasma
membrane (Lazarides and Burridge, 1975). In striated and
smooth muscle, a-actinin is most prominently concen-
trated in the Z discs and dense bodies and plaques, respec-
tively (Blanchard et al., 1989). Mutation of the gene en-
coding a-actinin in Drosophila results in disorganized
myofibrillar arrays and reduced muscle function (Fyrberg
et al., 1990; Roulier et al., 1992).
Prior to this report it was known that a-actinin has the
capacity to interact with four different adhesion plaque
and cytoskeletal proteins: integrin, vinculin, zyxin, and ac-
tin. A model for the associations among these proteins,
based on what has been learned from protein binding
studies, is shown in Fig. 11 B. Binding studies using the
proteolytic fragments of a-actinin digested by thermolysin
have shown that the b
1
integrin subunit interacts with the
53-kD rod-like domain of a-actinin (Otey et al., 1990),
whereas vinculin, zyxin, and actin interact with the 27-kD
globular head of a-actinin (Mimura and Asano, 1986;
Pavalko and Burridge, 1991; Crawford et al., 1992). Here
we have demonstrated an interaction between CRP1 and
the 27-kD globular head domain of a-actinin. We have
also shown that a-actinin interacts with the NH
2
-terminal
region of CRP1 (CRP1-LIM1) which contains one LIM
domain followed by a glycine-rich repeat. Moreover, by
heterologous protein expression experiments, we have
Figure 8. CRP1 interacts with the 27-kD
actin-binding site of a-actinin. (A) A Coo-
massie blue–stained gel showing molecu-
lar mass markers M, purified a-actinin
(lane 1), and the 53- and 27-kD proteolytic
products of a-actinin generated by ther-
molysin cleavage (lane 2). Autoradio-
graph of overlay assay performed on par-
allel nitrocellulose strips containing purified
a-actinin (lanes 19 and 10) and the pro-
teolytic fragments of a-actinin (lanes 29
and 20) probed with [
32
P]GST-CRP1 (B),
or [
32
P]GST (C). Note that in the experi-
ment shown, thermolysin cleavage of
a-actinin was not complete, therefore
products other than the 53- and 27-kD
fragments are also detected. (D) Autora-
diograph illustrating the quality of the
bacterially expressed, purified,
32
P-labeled
probes, [
32
P]GST-CRP1 and [
32
P]GST.
Figure 9. The binding site for a-actinin on CRP1 is contained
within the CRP1-LIM1 fragment. (A) Coomassie blue–stained
gel showing the purified CRP1 (lane 1), the purified CRP1-LIM1
fragment (lane 2) and the purified CRP1-LIM2 fragment (lane
3). 100 pmoles of CRP1, 200 pmoles of CRP1-LIM1, and 200
pmoles of CRP1-LIM2 were loaded on the gel. The positions of
CRP1, CRP1-LIM1, and CRP1-LIM2 are marked (CRP1, LIM1,
and LIM2, respectively). The corresponding blot overlay assay
probed with [
125
I]a-actinin is shown in B. (C) Autoradiograph il-
lustrating the purity of the radioiodinated a-actinin probe. The
position of the molecular mass markers is indicated on the left
in kD.

The Journal of Cell Biology, Volume 139, 1997 166
shown that the full-length CRP1 and the CRP1-LIM1 pep-
tide have the capacity to localize along the actin cytoskele-
ton whereas the COOH-terminal part of the protein does
not. These results illustrate that the sequence information
required both for a-actinin binding and the cytoskeletal
localization of CRP1 is contained within the NH
2
-terminal
107 aa of the protein. Based on these observations, we
speculate that it is CRP1’s ability to bind to a-actinin that
targets it to the actin cytoskeleton; however, additional
work will be necessary to demonstrate whether this is in-
deed the case. The primary features of the NH
2
-terminal
107 aa of CRP1 are the presence of a single LIM domain
and a glycine-rich repeat. Since LIM domains have been
shown to function in specific protein–protein interactions,
it seems likely that the COOH-terminal LIM domain of
CRP1 will also interact with a specific binding partner.
CRP1 could thus serve as an adaptor protein that is tar-
geted to the actin cytoskeleton by virtue of an interaction
with a-actinin. The COOH-terminal LIM domain of CRP1
may function as a ligand for a factor whose function de-
pends on a cytoskeletal localization (Fig. 11 B).
CRP family members appear to play a role in muscle
differentiation; however, the mechanism by which they
might act has not been clarified. The ability of CRP1 to in-
teract in vivo with a-actinin in smooth muscle cells raises
the possibility that a-actinin and CRP1 cooperate to per-
form an essential function in smooth muscle differentia-
tion. One intriguing possibility is that the two proteins col-
laborate to localize protein machineries involved in actin
assembly or dynamics (Fig. 11 B). It is interesting in this
Figure 10. CRP1-LIM1 is targeted to actin stress fibers. Expression constructs encoding myc-tagged CRP1 (A and B), CRP1-LIM1 (C
and D), or CRP1-LIM2 (E and F) were microinjected into rat embryo fibroblast (REF52) cells. Double-label immunofluorescence was
used to compare the subcellular distributions of the expressed CRP1 polypeptides (A, C, and E) and the actin stress fibers (B, D, and F).
The expressed proteins were visualized using an anti-myc monoclonal antibody whereas the actin stress fibers were visualized with phal-
loidin. Bar, 30 mm.

Pomiès et al. CRP1 Interacts with
a
-Actinin 167
regard that both a-actinin and CRPs bind zyxin, a protein
that has been implicated in the spatial control of actin as-
sembly by virtue of its ability to bind Ena/VASP family
members that associate with profilin (Reinhard et al., 1995
a, b; Gertler et al., 1996). In future work, it will be very im-
portant to perform immunocytochemical studies to char-
acterize the subcellular distributions of CRP1 and a-actinin
within intact tissues. Likewise, functional studies will be
essential in order to assess the physiological significance of
the CRP1–a-actinin interaction in vivo.
In summary, using in vitro and in vivo biochemical stud-
ies and immunochemistry we have demonstrated a direct
and specific interaction between the LIM domain protein
CRP1 and the cytoskeletal protein, a-actinin. Our data are
consistent with the possibility that the localization of
CRP1 along the actin cytoskeleton is due to the interac-
tion between the NH
2
-terminal LIM domain of CRP1 and
the actin-binding protein, a-actinin. CRP1 has been impli-
cated as a key regulator in the control of muscle differenti-
ation. The appropriate targeting of CRP1 to the actin cy-
toskeleton is likely to be important for the function of the
protein during myogenesis. Given the finding that loss of
one CRP family member, MLP/CRP3, results in dramatic
disorganization of myofibrils (Arber et al., 1997), it is rea-
sonable to speculate that the appropriate localization and
function of CRPs at subcellular domains that are enriched
in a-actinin may be required in order for a cell to build or
maintain the semicrystalline arrays of actin and myosin fil-
aments that constitute the contractile machinery.
The authors thank all members of the Beckerle laboratory for helpful dis-
cussions (University of Utah, Salt Lake City, UT). We are particularly
grateful to K.L. Schmeichel who generously provided the CRP1-LIM1
and CRP1-LIM2 expression constructs used in this study. Thanks go to E.
King for assistance and helpful discussions with confocal immunofluores-
cence microscopy. We thank K. Shepard and J.D. Pino for generating the
B37 antibody, K. Burridge (University of North Carolina, Chapel Hill,
NC) for his generous gift of a-actinin antibodies, and S.A. Liebhaber
(University of Pennsylvania School of Medicine, Philadelphia, PA) for
providing the GST-hCRP1 expression construct.
This research was supported by grants from the National Institutes of
Health and the American Cancer Society to M.C. Beckerle and from the
Philippe Foundation, Inc. to P. Pomiès. M.C. Beckerle is the recipient of a
Faculty Research Award from the American Cancer Society.
Received for publication 6 December 1996 and in revised form 2 July
1997.
References
Arber, S., and P. Caroni. 1996. Specificity of single LIM motifs in targeting and
LIM/LIM interactions in situ. Genes Dev. 10:289–300.
Arber, S., G. Halder, and P. Caroni. 1994. Muscle LIM protein, a novel essen-
tial regulator of myogenesis, promotes myogenic differentiation. Cell. 79:
221–231.
Arber, S., J.J. Hunter, J. Ross, M. Hongo, G. Sansig, J. Borg, J.-C. Perriard,
K.R. Chien, and P. Caroni. 1997. MLP-deficient mice exhibit a disruption of
cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart
failure. Cell. 88:393–403.
Beckerle, M.C. 1986. Identification of a new protein localized at sites of cell-
substrate adhesion. J. Cell Biol. 103:1679–1687.
Beckerle, M.C., and K.R. Porter. 1983. Analysis of the roles of microtubules
and actin in erythrophore intracellular transport. J. Cell Biol. 96:354–362.
Belkin, A.M., and V.E. Kotelianski. 1987. Interaction of iodinated vinculin,
metavinculin and a-actinin with cytoskeletal proteins. FEBS (Fed. Eur. Bio-
chem. Soc.) Lett. 220:291–294.
Blanchard, A., V. Ohanian, and D. Critchley. 1989. The structure and function
of a-actinin. J. Muscle Res. Cell Motil. 10:280–289.
Cossu, G., S. Tajbakhsh, and M. Buckingham. 1996. How is myogenesis initi-
ated in the embryo? Trends Genet. 12:218–223.
Crawford, A.W., and M.C. Beckerle. 1991. Purification and characterization of
zyxin, an 82,000-dalton component of adherens junctions. J. Biol. Chem. 266:
5847–5853.
Crawford, A.W., J.W. Michelsen, and M.C. Beckerle. 1992. An interaction be-
tween zyxin and a-actinin. J. Cell Biol. 116:1381–1393.
Crawford, A.W., J.D. Pino, and M.C. Beckerle. 1994. Biochemical and molecu-
lar characterization of the chicken cysteine-rich protein, a developmentally
regulated LIM-domain protein that is associated with the actin cytoskeleton.
J. Cell Biol. 124:117–127.
Feuerstein, R., X. Wang, D. Song, N.E. Cooke, and S.A. Liebhaber. 1994. The
LIM/double zinc-finger motif functions as a protein dimerization domain.
Proc. Natl. Acad. Sci. USA. 91:10655–10659.
Freyd, G., S.K. Kim, and R. Horvitz. 1990. Novel cysteine-rich motif and homeo-
domain in the product of the Caenorhabditis elegans cell lineage gene lin-11.
Nature (Lond.). 344:876–879.
Fyrberg, E., M. Kelly, E. Ball, C. Fyrberg, and M.C. Reedy. 1990. Molecular ge-
netics of Drosophila a-actinin: mutant alleles disrupt Z disc integrity and
muscle insertions. J. Cell Biol. 110:1999–2011.
Gertler, F.B., K. Niebuhr, M. Reinhard, J. Wehland, and P. Soriano. 1996.
Mena, a relative of VASP and Drosophila Enabled, is implicated in the con-
trol of microfilament dynamics. Cell. 87:1–20.
Gimona, M., M. Herzog, J. Vandekerckhove, and J.V. Small. 1990. Smooth
muscle specific expression of calponin. FEBS (Fed. Eur. Biochem. Soc.) Lett.
274:159–162.
Imamura, M., T. Endo, M. Kuroda, T. Tanaka, and T. Masaki. 1988. Substruc-
ture and higher structure of the chicken smooth muscle a-actinin molecule.
J. Biol. Chem. 263:7800–7805.
Karlsson, O., S. Thor, T. Norberg, H. Ohlsson, and T. Edlund. 1990. Insulin
gene enhancer binding protein Isl-1 is a member of a novel class of proteins
containing both a homeo- and a Cys-His domain. Nature (Lond.). 344:879–
882.
Kosa, J.L., J.W. Michelsen, H.A. Louis, J.I. Olsen, D.R. Davis, M.C. Beckerle,
and D.R. Winge. 1994. Common metal ion coordination in LIM domain pro-
teins. Biochemistry. 33:468–477.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature (Lond.). 227:680–685.
Lazarides, E., and K. Burridge. 1975. a-Actinin: immunofluorescent localiza-
tion of a muscle structural protein in nonmuscle cells. Cell. 6:289–298.
Louis, H.A., J.D. Pino, K.L. Schmeichel, P. Pomiès, and M.C. Beckerle. 1997.
Comparison of three members of the cysteine-rich protein (CRP) family re-
veals functional conservation and divergent patterns of gene expression. J.
Biol. Chem. In press.
Maruyama, K., and S. Ebashi. 1965. a-actinin, a new structural protein from
striated muscle. II. Action of actin. J. Biochem. (Tokyo). 58:13–19.
Michelsen, J.W., K.L. Schmeichel, M.C. Beckerle, and D.R. Winge. 1993. The
LIM motif defines a specific zinc-binding protein domain. Proc. Natl. Acad.
Sci. USA. 90:4404–4408.
Michelsen, J.W., A.K. Sewell, H.A. Louis, J.I. Olsen, D.R. Davis, D.R. Winge,
Figure 11. a-Actinin and its
binding partners. (A) A sum-
mary of the interactions be-
tween a-actinin and its bind-
ing partners: integrin, zyxin,
actin, vinculin, and CRP1.
References for the dissocia-
tion constant values are as
follows: a-actinin–integrin,
(Otey et al., 1990); a-actinin–
zyxin, (Crawford et al.,
1992); a-actinin–vinculin,
(Wachsstock et al., 1987);
a-actinin–actin, (Wachsstock
et al., 1993); a-actinin–CRP1,
this report; vinculin–actin,
(Menkel et al., 1994). (B) A
schematic representation of
an adhesion plaque showing
a model for the association of
a-actinin with its known
binding partners within a cell.
a-Actinin and CRP1 may co-
operate to localize a complex
of zyxin, Ena/VASP, and profilin and thus could participate in
the regulation of actin assembly dynamics. (Z) zyxin; (P) profilin;
(V) vinculin; (
a
-A) a-actinin; (X) other CRP1-binding partners;
(PM) plasma membrane; (ECM) extracellular matrix.

The Journal of Cell Biology, Volume 139, 1997 168
and M.C. Beckerle. 1994. Mutational analysis of the metal sites in a LIM do-
main. J. Biol. Chem. 269:11108–11113.
Mimura, N., and A. Asano. 1986. Isolation and characterization of a conserved
actin-binding domain from rat hepatic actinogelin, rat skeletal muscle, and
chicken gizzard a-actinins. J. Biol. Chem. 261:10680–10687.
Menkel, A.R., M. Kroemker, P. Bubeck, M. Ronsiek, G. Nikolai, and B.M.
Jockusch. 1994. Characterization of an F-actin-binding domain in the cyto-
skeletal protein vinculin. J. Cell Biol. 126:1231–1240.
Molkentin, J.D., and E.N. Olson. 1996. Defining the regulatory networks for
muscle development. Curr. Opin. Genet. Dev. 6:445–453.
Otey, C.A., F.M. Pavalko, and K. Burridge. 1990. An interaction between
a-actinin and the b
1
integrin subunit in vitro. J. Cell Biol. 111:721–729.
Pavalko, F.M., and K. Burridge. 1991. Disruption of the actin cytoskeleton after
microinjection of proteolytic fragments of a-actinin. J. Cell Biol. 114:481–
491.
Perez-Alvarado, G.C., C. Miles, J.W. Michelsen, H.A. Louis, D.R. Winge, M.C.
Beckerle, and M.F. Summers. 1994. Structure of the C-terminal LIM domain
from the cysteine rich protein, CRP. Nat. Struct. Biol. 1:388–398.
Podlubnaya, Z.A., L.A. Tskhovrebova, M.M. Zaalishvili, and G.A. Stefanenko.
1975. Electron microscopic study of a–actinin. J. Mol. Biol. 92:357–359.
Reinhard, M., K. Giehl, K. Abel, C. Haffner, T. Jarchau, V. Hoppe, B. M. Jock-
usch, and U. Walter. 1995a. The proline-rich focal adhesion and microfila-
ment protein VASP is a ligand for profilins. EMBO (Eur. Mol. Biol. Organ.)
J. 14:1583-1589.
Reinhard, M., K. Jouvenal, D. Tripier, and U. Walter. 1995b. Identification, pu-
rification, and characterization of a zyxin-related protein that binds the focal
adhesion and microfilament protein VASP (vasodilator-stimulated phos-
phoprotein). Proc. Natl. Acad. Sci. USA. 92:7956–7960.
Roulier, E.M., C. Fyrberg, and E. Fyrberg. 1992. Perturbations of Drosophila
a-actinin cause muscle paralysis, weakness, and atrophy but do not confer
obvious nonmuscle phenotypes. J. Cell Biol. 116:911–922.
Sadler, I., A.W. Crawford, J.W. Michelsen, and M.C. Beckerle. 1992. Zyxin and
cCRP: two interactive LIM domain proteins associated with the cytoskele-
ton. J. Cell Biol. 119:1573–1587.
Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Labo-
ratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
545 pp.
Schmeichel, K.L., and M.C. Beckerle. 1994. The LIM domain is a modular pro-
tein binding interface. Cell. 79:211–219.
Stronach, B.E., S.E. Siegrist, and M.C. Beckerle. 1996. Two muscle-specific
LIM proteins in Drosophila. J. Cell Biol. 134:1179–1195.
Wachsstock, D.H., W.H. Schwartz, and T.D. Pollard. 1993. Affinity of a-actinin
for actin determines the structure and mechanical properties of actin fila-
ment gels. Biophys. J. 65:205–214.
Wachsstock, D.H., J.A. Wilkins, and S. Lin. 1987. Specific interaction of vincu-
lin with a-actinin. Biochem. Biophys. Res. Commun. 146:554–560.
Wallraff, E., M. Schleicher, M. Modersitzki, D. Rieger, G. Isenberg, and G.
Gerisch. 1986. Selection of Dictyostelium mutants defective in cytoskeletal
proteins: use of an antibody that binds to the ends of a-actinin rods. EMBO
(Eur. Mol. Biol. Organ.) J. 5:61–67.
Weiskirchen, R., J.D. Pino, T. Macalma, K. Bister, and M.C. Beckerle. 1995.
The cysteine-rich protein family of highly related LIM domain proteins. J.
Biol. Chem. 270:28946–28954.